A novel control of human keratin expression: cannabinoid receptor 1-mediated signaling down-regulates the expression of keratins K6 and K16 in human keratinocytes in vitro and in situ

A novel control of human keratin expression: cannabinoid receptor 1-mediated signaling down-regulates the expression of keratins K6 and K16 in human keratinocytes in vitro and in situ https://t.co/W21MHvykf8 via @thePeerJ

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Abstract

Cannabinoid receptors (CB) are expressed throughout human skin epithelium. CB1 activation inhibits human hair growth and decreases proliferation of epidermal keratinocytes. Since psoriasis is a chronic hyperproliferative, inflammatory skin disease, it is conceivable that the therapeutic modulation of CB signaling, which can inhibit both proliferation and inflammation, could win a place in future psoriasis management. Given that psoriasis is characterized by up-regulation of keratins K6 and K16, we have investigated whether CB1 stimulation modulates their expression in human epidermis. Treatment of organ-cultured human skin with the CB1-specific agonist, arachidonoyl-chloro-ethanolamide (ACEA), decreased K6 and K16 staining intensity in situ. At the gene and protein levels, ACEA also decreased K6 expression of cultured HaCaT keratinocytes, which show some similarities to psoriatic keratinocytes. These effects were partly antagonized by the CB1-specific antagonist, AM251. While CB1-mediated signaling also significantly inhibited human epidermal keratinocyte proliferation in situ, as shown by K6/Ki-67-double immunofluorescence, the inhibitory effect of ACEA on K6 expression in situ was independent of its anti-proliferative effect. Given recent appreciation of the role of K6 as a functionally important protein that regulates epithelial wound healing in mice, it is conceivable that the novel CB1-mediated regulation of keratin 6/16 revealed here also is relevant to wound healing. Taken together, our results suggest that cannabinoids and their receptors constitute a novel, clinically relevant control element of human K6 and K16 expression.

Skin samples were first incubated overnight to adapt to culture conditions after which the medium was replaced and vehicle or test substances were added. For human skin organ culture, skin samples were treated with ACEA (Sigma-Aldrich, Taufkirchen, Germany, 30 µM) or AM251 (Sigma-Aldrich, 1 µM), or the combination of them for 1-day after the overnight incubation (Sugawara et al., 2012). Following culturing for the time indicated, skin samples were cryoembedded and prepared for histology, immunohistochemistry/immunofluorescence and quantitative immunohistomorphometry (Ramot et al., 2010; Ramot et al., 2011; Sugawara et al., 2012). Each evaluation was performed on 2–4 sections of 2 skin fragments per each treatment group from 2–4 individuals.

qRT-PCR

qRT-PCR was performed on an ABI Prism 7000 sequence detection system (Applied Biosystems/Life Technologies, Foster City, CA, USA) using the 5’ nuclease assay as detailed in our previous reports (Toth et al., 2011; Toth et al., 2009). Total RNA was isolated from HaCaT keratinocytes using TRIreagent (Applied Biosystems/Life Technologies, Foster City, CA, USA) and digested with recombinant RNase-free DNase-1 (Applied Biosystems) according to the manufacturer’s protocol. After isolation, one µg of total RNA was reverse-transcribed into cDNA by using High Capacity cDNA kit (Applied Biosystems) following the manufacturer’s protocol. PCR amplification was performed by using specific TaqMan primer and probes (Applied Biosystems, assay ID: Hs01699178_g1 for human K6A). As internal housekeeping gene control, transcripts of cyclophillin A (PPIA) were determined (Assay ID: Hs99999904 for human PPIA). The amount of the K6A transcripts was normalized to the control gene using the ΔCT method.

Immunohistochemistry

For the detection of K6 in organ cultured human skin as well as cultured HaCaT KCs, indirect immunofluorescence staining was performed using mouse anti-human K6 antibody (Progen, Ks6.KA12) at 1:10 dilution as a primary antibody and rhodamine conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA) at 1:200 dilution in phosphate-buffered saline (PBS) as a secondary antibody.

To study the proliferation of epidermal KCs, double-immunostaining for K6 and Ki-67 was performed. Briefly, after the staining for K6 with FITC conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratories) as a secondary antibody, sections were incubated overnight at 4 °C with a mouse anti-human Ki-67 antibody (DAKO, Hamburg, Germany) at 1:20 in PBS. Sections were then washed with PBS, followed by incubation with rhodamine conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratories) (1:200 in PBS, 45 min) at room temperature.

For all immunostainings, the respective primary antibodies were omitted as negative controls, and morphological criteria and reproduction of the previously published intracutaneous expression patterns of the examined antigens were used as internal positive and negative controls (Moll, Divo & Langbein, 2008). For all experiments, control and treated sections were stained (and later evaluated) on the same day by the same investigator. To avoid staining biases, we calculated the relative staining intensity (arbitrary intensity; 1 as control group) among treatment groups per each individual and then pooled data from all of the experiments.

The staining intensity of K6 and K16 in defined reference areas was assessed by quantitative immunohistomorphometry using the ImageJ software (NIH: National Institutes of Health, Bethesda, MD) (Bodo et al., 2010; Ramot et al., 2010; Ramot et al., 2011). For epidermal evaluation, staining intensity was evaluated in the suprabasal cells, and for HaCaT cells, staining intensity was measured in the colonies formed. For K6 immunofluorescence intensity, skin sections from 4 different individuals were used, while K16 immunofluorescence intensity and %Ki67 were evaluated in skin sections from 2 patients. 4–6 sections per one individual (2 sections per investigated skin fragment) were used for each evaluation. Each section was evaluated in two/three different non-adjacent microscopic fields (×200), and the mean intensity was measured, and considered as a value. Each treatment group was compared to the control group (average value), and relative change in expression was calculated. Highly comparable results were obtained from different sections from different individuals.

Statistical analysis

Significance of difference between two groups was evaluated using Student’s t-test for unpaired samples. For multiple comparisons, one-way analysis of variance (ANOVA) was used, followed by Bonferroni’s multiple comparison test, using Prism 5.0 software (GraphPad Prism Program, GraphPad, San Diego, CA). p values <0.05 were regarded as significant. All data in the figures are expressed as mean + SEM. *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated comparisons.

Results and discussion

First, we asked whether the CB1-specific synthetic agonist ACEA (Pertwee et al., 2010; Sugawara et al., 2012) can modulate the expression of keratin K6 in human skin. K6 staining intensity within the epidermis of full-thickness human skin that had been organ-cultured for 24 h under serum-free conditions in the presence of ACEA (30 µM) or vehicle alone was assessed by quantitative immunohistomorphometry. This showed that K6 immunoreactivity (IR) was significantly reduced after ACEA treatment, compared to the vehicle control group (Figs. 1A and 1B).

This down-regulation was abrogated in part by the co-administration with the CB1-specific antagonist, AM251 (Pertwee et al., 2010; Sugawara et al., 2012) (Figs. 1A and 1B). Therefore, intraepidermal K6 protein expression in normal human skin in situ is down-regulated in a CB1-specific manner.

ACEA also down-regulates K16 protein expression in situ

Since K16 is the type I keratin partner of K6 in KCs and is thought to stabilize this keratin protein as a cytoskeletal heteropolymeric intermediate filament (Ramot et al., 2009), we next analyzed K16 IR in the epidermis of organ cultured human skin samples treated with ACEA. In line with the K6 protein expression, K16 IR was also significantly down-regulated by ACEA in situ (Figs. 1C and 1D).

Just as we had seen before with the non-selective endocannabinoid, AEA (Toth et al., 2011), the CB1-specific synthetic agonist, ACEA indeed significantly decreased human epidermal KC proliferation in situ. This effect was abrogated by the CB1-specific antagonist, AM251 (assessed by quantitative Ki-67 immunomorphometry, Figs. 3A and 3B).

Therefore, it needed to be dissected whether or not CB1 also reduces K6 expression in a proliferation-independent manner. This was done by selectively assessing K6 expression in non-proliferative (i.e. Ki-67-negative) epidermal KCs in situ. We found that K6 IR within non-proliferative epidermis was also reduced by ACEA (Figs. 4A and 4B). Furthermore, K6-expressing cells in the epidermis co-expressed CB1 in situ (Fig. 4C), suggesting a direct effect of CB1-agnosits on K6-expressing human epidermal KCs in situ. Thus, CB1 stimulation may affect K6 expression both, by reducing KC proliferation and by down-regulating K6 expression directly via CB1 in a proliferation-independent manner.

Here we provide the first evidence that CB1-mediated signaling directly regulates K6/16 expression within normal human skin. Specifically, we show that CB1 stimulation down-regulates expression of the hyper-proliferation-associated human keratins K6 in vitro and in situ, and inhibits human epidermal KC proliferation in situ.

The effect of CB-mediated signaling in human KC biology remains to be fully explored. As we have also observed in isolated human skin (Fig. 4C), CB1 protein expression is detected mainly above the basal layer of the epidermis (Stander et al., 2005), i.e. above the compartment where KC proliferation normally occurs most prominently. Wilkinson and Williamson reported that the non-selective CB agonist HU210 inhibited KC proliferation. However, this could not be blocked by either CB1 or CB2 antagonists, suggesting that cannabinoids may also inhibit human KC proliferation through a non-CB1/CB2 mechanism (Wilkinson & Williamson, 2007). Nevertheless, it has been previously shown that AEA, which can interact with CB1 on human KC (Biro et al., 2009), inhibited human KC proliferation in situ and in vitro (Toth et al., 2011).

It should be noted, that the constitutive level of K6 expression in organ-cultured human skin fragments is considerably higher than normal scalp skin in vivo. Presumably, this occurs as a response to tissue dissection and organ culture, which is well-known to elicit an immediate wound healing response in the epithelium. The latter rapidly starts to migrate over the wound edge in an attempt to enwrap the exposed skin mesenchyme (epiboly phenomenon Stenn, 1981; Brown et al., 1991). This constitutive up-regulation of K6 in organ-cultured normal human skin may greatly heighten the sensitivity to K6 expression-modulatory compounds, such as CB ligands, thus making human skin organ culture a particularly sensitive and instructive tool for clinically relevant keratin research. At the same time, however, caution is advised in extrapolating from these observation made in what essentially reflects a wound healing milieu to healthy, unmanipulated human skin in vivo.

The current findings invite the speculation that the therapeutic down-modulation of K6 and/or K16 expression by CB1 agonists and other cannabimimetics might become exploitable for the management of other dermatoses besides psoriasis, for example pachyonychia congenita (Hickerson et al., 2011; Zhao et al., 2011) and acne (Biro et al., 2009), and could be used to modulate KC migration-dependent reepithelialization in wound healing, similar to related findings in periodontal and intestinal wound repair (Kozono et al., 2010; Wright et al., 2005).

Conclusion

Our results suggest that cannabinoids and their receptors constitute a novel, clinically relevant control element of human K6 and K16 expression. Therefore, cannabimimetic agents might be relevant for the treatment of several skin conditions related to aberrant K6/K16 expression, such as psoriasis and wound healing. In addition, skin organ culture is shown to be a clinically and physiologically relevant model system for investigating the effect of CB1 specific agonists/antagonists on human skin.

Abbreviations

ACEA

arachidonoyl-chloro-ethanolamide

AEA

anandamide

CB1

cannabinoid receptor 1

ECS

endocannabinoid system

HF

hair follicle

KC

keratinocyte

Acknowledgements

The authors thank Motoko Sugawara for her excellent technical assistance, and Dr. Stephan Tiede for professional advice. The generous professional support of Prof. Masamitsu Ishii and Prof. Hiromi Kobayashi, Osaka, for this work is also gratefully appreciated. Dr. Wolfgang Funk is gratefully acknowledged for harvesting human skin samples for the organ culture.

Additional Information and Declarations

Competing Interests

Ralf Paus is an Academic Editor for PeerJ.

Author Contributions

Yuval Ramot and Koji Sugawara conceived and designed the experiments, performed the experiments, analyzed the data, wrote the paper.

Ralf Paus conceived, designed, and supervised the experiments, and wrote the paper.

Human Ethics

The following information was supplied relating to ethical approvals (i.e. approving body and any reference numbers):

Institutional review board ethics committee of the University of Luebeck, reference number 06-109.

Funding

This study was supported in part by the Daniel Turnberg United Kingdom/Middle East Travel Fellowship Scheme, administered by the Academy of Medical Sciences to YR; a faculty grant from Osaka City University to KS; a “Lendület” grant of the Hungarian Academy of Sciences to TB; and a faculty grant from the University of Luebeck to RP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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